Affective disorders, such as bipolar disorder, schizophrenia and PTSD, as well as drug and alcohol addiction, are increasingly being understood as dysfunctions of specific brain circuits. In order to develop new, rational therapeutic approaches to these illnesses, it is necessary to understand the normal function of the disrupted circuits, and how they are altered in a given disorder. Central to this objective is the use of molecular genetic-based tools, in the mouse, to mark, map and manipulate neural circuitry. In this application, we propose to develop new, genetically based, techniques for mapping the connectivity of neurons that are activated by a specific stimulus or during a specific behavior. Specifically, our goal is to fill two lacunae in the current toolkit for genetic mapping of neuronal circuits in mice: a conditional, viral-based method for anterograde trans-neuronal tracing; and a method to stably and efficiently mark neurons that have been transiently activated. In Specific Aim I, we will develop recombinant variants of the Herpesvirus strain H129, which is transported trans-neuronally in the anterograde direction, using a homologous recombination-based method we have developed. These variants will be dependent on Cre recombinase for expression of a marker and/or replication. We will test these recombinant strains in several lines of Cre-expressing mice, in both the peripheral and central nervous system. In Specific Aim II, we will develop and test two methods for stable, activity-dependent marking (SADM) of neurons, both of which are based on the expression of Cre recombinase. These methods are based on the combined use of transgenic mice and stereotaxically injected, Cre-dependent recombinant viruses. They allow, in principle, the expression of any marker or effector gene in transiently activated neurons. In Specific Aim III, we will combine the technologies of Aims I and II, to achieve activity-dependent retrograde and anterograde trans-neuronal tracing of neural circuits. Finally, in Specific Aim IV, we will modify the methods of Aim II, to permit selective stable, activity-dependent marking of GABAergic neurons (SADM-GAD). This will permit the visualization of active inhibitory networks in the brain, and will open the way to marking and manipulating them in an activity-dependent manner. The methods described in this application will make available to the community a new set of tools that should have wide applications in neuronal circuit tracing, in both normal mice and disease models. PUBLIC HEALTH RELEVANCE: The development of new treatments for psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), as well as for drug and alcohol addiction, will require a better understanding of the brain circuits whose functions are disrupted in these disorders. The present application is aimed at developing new, molecular genetically based, methods for marking, mapping and manipulating neural circuits in the mouse, the best genetically accessible system for modeling such disorders. These methods should have wide applicability in furthering our understanding of the neural circuit basis of affective disorders and addiction in this important model system, and may identify new cellular targets for the development of novel therapeutics.